CN110265089B - Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof - Google Patents
Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof Download PDFInfo
- Publication number
- CN110265089B CN110265089B CN201910538333.2A CN201910538333A CN110265089B CN 110265089 B CN110265089 B CN 110265089B CN 201910538333 A CN201910538333 A CN 201910538333A CN 110265089 B CN110265089 B CN 110265089B
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- amplification reaction
- quantitative analysis
- mrna
- color
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B45/00—ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medical Informatics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Theoretical Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Data Mining & Analysis (AREA)
- Bioinformatics & Computational Biology (AREA)
- Wood Science & Technology (AREA)
- Evolutionary Biology (AREA)
- Databases & Information Systems (AREA)
- Software Systems (AREA)
- Public Health (AREA)
- Evolutionary Computation (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Bioethics (AREA)
- Artificial Intelligence (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a nucleic acid quantitative analysis method based on intelligent equipment assistance and application thereof, wherein the method is characterized in that a nucleic acid amplification reaction system sensitive to pH is initiated by a nucleic acid target object through design and construction, and visual rapid analysis of nucleic acid by naked eyes is realized by adopting a pH indicator cresol red with a color change range of yellow 7.2-pink 8.8; on the basis, a three-primary-color image scanning mode assisted by intelligent equipment is utilized, the intelligent equipment detection terminal is used for realizing rapid detection and quantitative analysis of nucleic acid, and the used intelligent equipment comprises intelligent equipment with a photographing function, such as a smart phone and a tablet.
Description
Technical Field
The invention belongs to the technical field of biological analysis, and particularly relates to a nucleic acid quantitative analysis method based on intelligent equipment assistance and application thereof+The method utilizes App in intelligent equipment (mobile phone, flat panel and the like) to read color information, namely three primary colors (red, green and blue) of an image of a nucleic acid amplification reaction system, establishes a relation between the three primary colors and the concentration of the nucleic acid, and realizes simple, convenient, rapid, high-sensitivity and high-specificity quantitative analysis on the nucleic acid.
Background
The simple, rapid, high-sensitivity and high-specificity nucleic acid detection and quantitative analysis play an important role in the fields of pathogenic microorganisms, DNA/RNA viruses, genetic disease diagnosis, food safety and the like.
At present, researchers amplify target DNA/RNA by using Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP) and Strand Displacement Amplification (SDA) amplification technologies, and establish a visual nucleic acid amplification product detection system by using a pH indicator, (BioTechniques,2015,58,59-68.) identify the 'positive or negative' of the target DNA/RNA through color changes before and after amplification reaction, although the simple and rapid detection of the target DNA/RNA is realized, the quantitative analysis cannot be realized. Plum et al, using PCR, Rolling Circle Amplification (RCA) and SDA amplification in combination with a pH indicator, achieve quantitative detection of miRNA by measuring the amplification product uv-vis spectrum, despite this, requires additional handling and measurement steps, is prone to contamination of the amplification product, and also requires expensive instrumentation.
The smart phone not only becomes a necessary communication tool in daily life, but also provides a high-performance photographing function for people. The visual nucleic acid detection result can be conveniently and quickly recorded by using the photographing function of the smart phone; in addition, the smart phone provides an operating environment for mobile phone software (App), and there are many open-source apps such as Pixel Picker, Color Picker, PickColor, etc. which can quickly read information of three primary colors (red, green, blue) of a shot photo. Based on image three primary colors (red, green and blue) analysis App, a smartphone is utilized to identify the color of a nucleic acid amplification reaction, and the relationship between the three primary colors and the concentration of nucleic acid is established, so that the quantitative analysis of the nucleic acid, which is simple, convenient, rapid, high in sensitivity and high in specificity, is realized, and a new tool is provided for molecular diagnosis analysis, disease diagnosis analysis and the like which take nucleic acid molecules as target objects.
Disclosure of Invention
The invention provides a nucleic acid quantitative analysis method based on intelligent equipment assistance, which utilizes App (intelligent mobile phone or tablet personal computer and the like) in intelligent equipment to quickly identify and read information of three primary colors (namely RGB numerical values) of a pH-sensitive nucleic acid amplification reaction system initiated by a nucleic acid target; the good linear relation between the normalized RGB value (G/(R + G + B)) and the negative logarithm of the concentration of the nucleic acid target is found for the first time, so that the simple and rapid nucleic acid quantitative analysis method is established by using intelligent equipment.
The technical scheme of the nucleic acid quantitative analysis method comprises the following steps:
1. according to the nucleic acid sequence to be detected, a pH-sensitive nucleic acid amplification system initiated by a nucleic acid target is designed and constructed, wherein the nucleic acid amplification system can be any one of the nucleic acid amplification reactions based on DNA polymerase, such as Polymerase Chain Reaction (PCR), rolling circle amplification Reaction (RCA), loop-mediated isothermal nucleic acid amplification reaction (LAMP), ligation-mediated polymerase chain reaction (L-PCR), ligation-mediated isothermal nucleic acid amplification reaction (LIEXA) and the like which have been developed at present.
2. Monitoring the nucleic acid amplification reaction with a pH indicator, wherein the nucleic acid amplification reaction generates H as the nucleic acid amplification reaction continues+Causing the color of the pH indicator to change, and under the appropriate amplification reaction time, identifying the nucleic acid target object by naked eyes through the color change of an amplification reaction system; meanwhile, the image of the color change of the reaction system can be conveniently recorded by utilizing the photographing function of the intelligent equipment, and the three primary colors RGB information of the image is obtained through the App in the intelligent equipment.
3. And fitting the RGB information and the nucleic acid concentration to obtain a linear relation between the RGB value and the nucleic acid concentration.
4. The linear relationship is used to perform quantitative analysis on nucleic acid.
Further, an mRNA quantitative analysis application based on an intelligent device assisted nucleic acid quantitative analysis method, the mRNA quantitative analysis application comprising:
s1: constructing a pH-sensitive, mRNA-initiated, ligation-dependent isothermal nucleic acid amplification reaction system;
s2: at different reaction times, shooting and recording the color of the amplification reaction by using intelligent equipment;
s3: acquiring RBG color information of the picture in S2 through the image processing App;
s4: constructing a linear relation between the RGB value in the color information and the nucleic acid concentration;
s5: based on the linear relationship in S4, mRNA in the sample to be determined is quantified using an intelligent device.
Further, the linear correlation in S4The equation is: G/(R + G + B) ═ 0.5078+0.0166lgCe13a2(M),[100pM-100fM，R2＝0.9943]；
G/(R+G+B)＝0.7222+0.0331lgCe13a2(M)[100fM-100aM，R2＝0.9979]。
Further, the S1 constructing an isothermal nucleic acid amplification reaction system includes:
s11: the detection recognition area of the probe SLP-e13 and the detection recognition area of the probe SLP-a2 are respectively hybridized with the mRNA of the transcript in the system specifically;
s12: using mRNA as a template, and connecting the probe SLP-e13 and the probe SLP-a2 by SplintR ligase to form double-stem ring structure DNA;
s13: and (3) initiating an amplification reaction by taking the DNA with the double-stem-loop structure as an initial template of the loop-mediated isothermal amplification reaction.
The invention has the following beneficial effects:
1. compared with the traditional nucleic acid quantitative analysis, the invention takes the smart phone, the panel and the like as the detection terminals, does not need expensive quantitative analysis instruments and has low analysis cost;
2. the method does not need additional operation steps, only needs to photograph the reaction tube with a smart phone, a tablet and the like after the reaction is finished, and analyzes the color information of the photograph, so that the quantitative analysis of the nucleic acid can be realized, and the method is simple and convenient to operate;
3. the invention takes the smart phone, the tablet and the like as the detection terminals, and is very suitable for clinical quick detection and household diagnosis analysis and detection;
4. the present invention is not limited to the quantitative analysis of mRNA as set forth in the examples, and can be generalized to the quantitative analysis of all nucleic acids based on amplification systems.
Drawings
FIG. 1 is a diagram of a standard mRNA quantitative determination curve for a BCR-ABL fusion gene established in the present invention;
FIG. 2 is a schematic diagram of a Fusion gene analysis scheme based on Vis-Fusion LIEXA method in accordance with an embodiment of the present invention;
FIG. 3 is a graph showing the color change of each reaction tube for different reaction times according to an embodiment of the present invention;
FIG. 4 is a graph showing the variation trend of the R values of different concentrations of mRNA reaction tubes after different amplification reaction times according to the embodiment of the present invention;
FIG. 5 is a graph showing the trend of the G value change of mRNA reaction tubes with different concentrations after different amplification reaction times according to the embodiment of the present invention;
FIG. 6 is a graph showing the trend of the B value of different concentrations of mRNA reaction tubes after different amplification reaction times according to the embodiment of the present invention;
FIG. 7 is a graph showing the trend of the change in G/(R + G + B) value of mRNA reaction tubes with different concentrations after different amplification reaction times according to the embodiment of the present invention;
FIG. 8 is a diagram showing the study of the specificity of the assay in accordance with the embodiment of the present invention;
FIG. 9 is a graph showing the results of quantitative detection of RNA in different cells according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. On the contrary, the invention is intended to cover alternatives, modifications, equivalents and alternatives which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention. It will be apparent to one skilled in the art that the present invention may be practiced without these specific details.
The invention is further described with reference to the following figures and specific examples, which are not intended to be limiting. The following are preferred examples of the present invention:
as shown in fig. 1 to 9, the present invention provides a RNA quantitative analysis method assisted by an intelligent device (mobile phone, tablet, etc.) and an application thereof, the method implements a visual rapid analysis of fusion genes by naked eyes by designing and constructing a pH-sensitive ligation-LAMP amplification system and using a pH indicator (such as cresol red, with a color change range of (yellow) 7.2 to 8.8 (pink)), and on this basis, a three-primary-color image scanning mode assisted by the intelligent device (mobile phone, tablet, etc.) is used to implement a rapid detection and quantitative analysis of fusion genes by a detection terminal of the intelligent device (mobile phone, tablet, etc.), the method of the present invention is abbreviated as: Vis-Fusion LIEXA; and the method is applied to the detection of the fusion gene in an actual sample.
The method takes intelligent equipment (mobile phone, flat panel and the like) as a carrier for recording the reaction color of nucleic acid, and H generated in the amplification reaction of the nucleic acid+Recording pH color change, reading three primary color values, establishing a fitting relation between the three primary color values and the nucleic acid concentration, carrying out nucleic acid quantitative analysis on the nucleic acid reaction color recorded by an intelligent device (a mobile phone, a flat plate and the like) through the fitting relation, and carrying out amplification reaction on the nucleic acid through H generated in the amplification reaction+The pH color change recording is specifically carried out by constructing an isothermal nucleic acid amplification system which is connected sensitively to pH, so that H + ions are generated in the nucleic acid amplification reaction process enough to cause the pH change of the system, the color of the cresol red pH indicator is changed from pink to yellow through the change, the existence of the fusion gene is recognized by naked eyes due to the color change, and the fitting relation between the three primary color numerical value and the nucleic acid concentration is a linear relation between the ratio of G/(R + G + B) and the negative logarithm of the mRNA concentration. Wherein the linear correlation equations between the ratio of G/(R + G + B) and the negative logarithm of the mRNA concentration are respectively.
The method comprises the following steps:
s1: based on the existing DNA/RNA nucleic acid amplification system based on the pH indicator, the time of the amplification reaction is optimized;
s2: shooting and recording the color of the amplification reaction by using intelligent equipment (a mobile phone, a tablet and the like);
s3: acquiring color information of each reaction tube in the photo through an image processing App;
s4: establishing a fitting relation between RGB values in the color information and the nucleic acid concentration;
s5: based on the fitting relationship in S4, the nucleic acid reaction color is photographed by an intelligent device (a mobile phone, a tablet, etc.) and the color information is read, so that quantitative analysis of the nucleic acid is realized.
The method for optimizing the amplification reaction time in S1 is as follows:
s11: the detection recognition area of the probe SLP-e13 and the probe SLP-a2 is specifically hybridized with the mRNA of the target transcript respectively;
s12: mRNA was used as template, SplintR ligase, T4RNA ligase 2 or other RNA ligase connects the probe SLP-e13 and the probe SLP-a2 to form double-stem-loop structure DNA;
s13: and (3) taking the DNA with the double-stem-loop structure as an initial template of isothermal exponential amplification reaction to initiate the amplification reaction.
The mRNA of the transcript of the nucleic acid in the S11 system needs to be quantitatively detected before specific hybridization, and the quantitative detection method comprises the following steps: adding 2.0 μ L nuclease-free water, 1.0 μ L2 nmol/L probe SLP-e13 water solution, 1.0 μ L2 nmol/L probe SLP-a2 water solution, 5.0 μ L2 Xligation reaction mixture, 1.0 μ L mRNA fragment water solution or RNA sample annealed at 75 deg.C for 2min into 200 μ L centrifuge tube, mixing uniformly, maintaining at 25 deg.C for 20min to complete ligation reaction, and replacing mRNA sample with 1.0 μ L water to make blank control experiment, wherein the 5.0 μ L2 Xligation reaction mixture is 20mM MgCl22mM ATP, 20mM DTT, 5units SplintR ligase and 100. mu.M Tris-HCl, pH 8.5@25 ℃.
The base sequence of the probe SLP-e13 is as follows:
5′-CTTCCTTATTGATGGTCAGCTTTATCGTCGTGACTGTTTGTAATAGGAC A GAGCCCCGCACTTTCAGTCACGACGAT-3′；
the base sequence of probe SLP-a2 is:
5′-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGTTTTACTGGCCGCTGAAGGGCTT-3′；
wherein the 5' end of the probe SLP-a2 is modified with phosphate group.
The isothermal exponential amplification reaction was performed at 65 ℃ for 16 minutes.
Example (b): BCR-ABL fusion gene mRNA analysis
The method comprises the steps of taking a molecular marker detected in clinical diagnosis, treatment and prognosis of Chronic Myelogenous Leukemia (CML) and BCR-ABL fusion gene mRNA as an RNA detection model, designing and synthesizing DNA probes SLP-e13 and SLP-a2 with stem-loop structures according to a patent granted previously (patent number: ZL201610316771.0), wherein the base sequence of the probe SLP-e13 is 5'-CTTCCTTATTGATGGTCAGCTTTATCGTCGTGACTGTTTGTAATAGGACAGAGCCCCGCACTTTCAGTCACGACGAT-3' (provided by Takara bioengineering (Dalian) Co., Ltd.);
the base sequence of the probe SLP-a2 was 5'-CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGATTTTCCTCTGCTGTCGTTTTACTGGCCGCTGAAGGGCTT-3' (provided by Takara Bio Inc.), wherein the 5 ' -end of the probe SLP-a2 was modified with a phosphate group.
As shown in fig. 2, according to the BCR-ABL Fusion gene mRNA sequence, two specific stem-loop structure DNA probes SLP-e13 and SLP-a2 were designed at the BCR gene and ABL gene Fusion site (Fusion junction), sequences capable of specifically recognizing exon 13 of BCR gene and exon 2 of ABL gene were included on SLP-e13 and SLP-a2 probes, respectively, and primer sequences required for isothermal index amplification (IEXA) were included on both probes; in the presence of a BCR-ABL e13a2 fusion gene mRNA molecule, which specifically hybridizes to the probe, followed by ligation of DNA probes using mRNA as a template by splntr ligase to form a double stem-loop structured DNA template, the double stem-loop structure can trigger rapid and efficient IEXA amplification in the presence of primers, dNTPs, and DNA polymerase, similar to the exponential amplification of a loop-mediated isothermal nucleic acid amplification reaction.
In the isothermal exponential amplification reaction, the DNA polymerase takes a molecule to be amplified (Template) as a Template and dNTP as a raw material, and the dNTP molecule is doped into the 3' end of the DNA to form a phosphodiester bond and release a hydrogen ion (H)+) When the reaction is carried out in a solution of low buffer capacity, H is produced+Enough to cause the system pH change, Jonathan Rothberg utilizes the principle to design an Ion Torrent semiconductor high-throughput sequencing system (Nature,2011,475, 348-. In the invention, H is generated in the nucleic acid amplification reaction process by constructing a pH-sensitive connection-isothermal nucleic acid amplification system+The ions are sufficient to cause a change in the pH of the system, which can cause the cresol red pH indicator to change color from pink to yellow, thereby turning onIdentifying the existence of the fusion gene by naked eyes through color change; in addition, the result is photographed by using intelligent equipment (a mobile phone, a tablet and the like), three primary colors RGB (Red, Green and Blue) values of the picture are read by using an image processing App, and a quantitative relation between the RGB values and the concentration of the fusion gene is researched and established, so that the detection and quantitative analysis of the fusion gene are realized.
The specific method for quantitatively detecting the mRNA of the BCR-ABL fusion transcript by adopting the strategy is as follows:
into a 200. mu.L centrifuge tube were added 2.0. mu.L of nuclease-free water, 1.0. mu.L of an aqueous solution of 2nmol/L probe SLP-e13, 1.0. mu.L of an aqueous solution of 2nmol/L probe SLP-a2, and 5.0. mu.L of a2 Xligation reaction mixture (20mM MgCl. sub.22mM ATP, 20mM DTT, 5units SplintR ligase and 100. mu.M Tris-HCl, pH 8.5@25 ℃), 1.0. mu.L mRNA fragment aqueous solution or RNA sample annealed at 75 ℃ for 2min, mixing uniformly, and keeping at 25 ℃ for 20min to complete the ligation reaction. At the same time, a blank control experiment was performed using 1.0. mu.L of water instead of the mRNA sample.
Subsequently, 2.0. mu.L of the ligation reaction product was added to 18.0. mu.L of a mixed solution for the loop-mediated isothermal amplification reaction consisting of 8. mu.L of nuclease-free water, 10.0. mu.L of a 2X hot start chromogenic-loop-mediated isothermal amplification reaction mixture (100mM KCl, 10mM (NH)4)2SO4，10mM MgSO45mM dNTPs, 0.2% Triton X-100, 12units Bst WarmStart 2.0DNA polymerase, 200. mu.M cresol red and 50. mu.M Tris-HCl, 1.2. mu.M Universal primer UP1(CGACAGCAGAGGATTTGTTGTGTGGAAGTGTGAGCGGA, supplied by Takara Bio Inc.), 1.2. mu.M Universal primer UP2(ATCGTCGTGACTGTTTGTAATAGGACAGAGCCC CG CAC, supplied by Takara Bio Inc.). After mixing uniformly, performing loop-mediated isothermal amplification reaction at 65 ℃, after amplifying for 16 minutes, photographing by using equipment (mobile phone, flat plate, etc.) to record color change. The intelligent device (mobile phone, flat panel, etc.) App is used for reading RGB values of mRNA samples with different concentrations after reaction, and a linear relation curve between the ratio of G/(R + G + B) and the negative logarithm of the target mRNA concentration is drawn, and the result is shown in figure 1.
The color of the system changes more obviously after the amplification reaction along with the increase of the concentration of mRNAAnd the different concentration gradients can be distinguished from each other, and at the same time, it can be seen from fig. 1 that the ratio of G/(R + G + B) and the negative logarithm of the mRNA concentration have a good linear relationship, and the linear correlation equations are G/(R + G + B) ═ 0.5078+0.0166lgCe13a2(M), [100pM-100fM, R2＝0.9943]And G/(R + G + B) 0.7222+0.0331lgCe13a2(M) [100fM-100aM, R2＝0.9979]. Therefore, the method of the present invention can quantitatively detect the target mRNA, and similarly, the method of the present invention can also be used for the quantitative detection of DNA.
The results of the above examples are as follows:
1. the data for optimizing amplification reaction time is shown in FIG. 3, in which FIG. 3 is a photograph of each reaction tube corresponding to different reaction times, the mRNA concentration in each image is sequentially 100pM,10pM,1pM,100fM,10fM,1fM,100aM, and 0 from left to right, and since the image is a black-and-white image, the color difference change cannot be seen from the image, the color in the image is darker red, and the color in the image is lighter yellow.
2. As shown in FIGS. 4-7, the trend of the change of RGB values of the mRNA reaction tubes with different concentrations after different amplification reaction times; in the quantitative analysis, the result is represented by a normalized RGB (G/(R + G + B)) value, and the representation mode can effectively eliminate quantitative errors caused by different photographing conditions.
3. The quantitative curve is shown in figure 1, according to the steps in the specific embodiment, the isothermal amplification reaction time (16min) in the Vis-Fusion LIEXA system is optimized, the performance of the method is examined by using the optimal conditions, and as can be seen from figure 3, the mRNA of the Fusion gene of BCR-ABL with the activity as low as 100aM can be visually detected by using the method; meanwhile, an intelligent device (mobile phone, flat panel and the like) assisted RGB value reading mode is utilized to find that a good linear relation exists between the G/(R + G + B) ratio and the concentration of the BCR-ABL fusion gene mRNA, so that the method can accurately and quantitatively detect the BCR-ABL fusion gene mRNA in a wide linear range.
4. Furthermore, we examined the specificity of the method, and as shown in FIGS. 8 and 9, the simultaneous detection of 100fM e1a2, e14a2, e19a2 and e13a2, which have homologous sequences to BCR-ABL e13a2, with stem-loop DNA probes SLP-e13 and SLP-a2 specific to e13a2, resulted in a significant color change only for e13a2, as shown in FIG. 8, indicating that the method can specifically detect the target fusion gene transcript. Meanwhile, the amplified products are analyzed by non-denaturing polyacrylamide gel electrophoresis, as shown in FIG. 9, only e13a2 generates a trapezoidal IXEA amplification product band, and other transcripts do not generate corresponding amplification products, which indicates that the Vis-Fuison LIEXA method completely depends on IEXA amplification initiated by ligation reaction products taking a target strand as a template. Further proves that the method has excellent specificity in the aspect of detecting the fusion gene. Finally, the method is successfully applied to the detection and quantitative analysis of BCR-ABL fusion genes in Total RNA samples.
The above-described embodiment is only one of the preferred embodiments of the present invention, and general changes and substitutions by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.
Claims (5)
1. A nucleic acid quantitative analysis method based on intelligent equipment assistance is characterized in that: the method comprises the steps of shooting and recording a picture of the color of a nucleic acid amplification reaction system initiated by a nucleic acid target object by using an intelligent device, then obtaining three primary colors of numerical values of nucleic acid amplification reaction in the picture, establishing a linear relation between the three primary colors of numerical values and the concentration of nucleic acid, and carrying out quantitative analysis on the nucleic acid by using the intelligent device through the linear relation, wherein the amplification reaction system is a pH sensitive nucleic acid amplification system, and H is generated in the amplification reaction process+Causing a change in the pH of the system, the change causing a color change in the pH indicator, and thereby the color change identifying the presence of the nucleic acid target to the naked eye, the smart device being configured to capture a record of the color of the nucleic acid amplification reaction and read the three primary color RGB values of the nucleic acid amplification reaction by App in the smart device, the read three primary color RGB values of the nucleic acid amplification reaction including R, G, B values or normalized B/(R + G + B), G/(R + G + B), and R/(R + G + B); the error generated by different shooting recording conditions in nucleic acid quantitative analysis is eliminated by normalized trichromatic numerical values of B/(R + G + B), G/(R + G + B) or R/(R + G + B), and linear correlation equations are respectively G/(R + G + B) ═ 0.5078+0.0166lgCe13a2 (R + G + B) ((R + G + B))M),[100pM-100fM，R2＝0.9943]And G/(R + G + B) 0.7222+0.0331lgCe13a2(M) [100fM-100aM, R2＝0.9979]。
2. The method for quantitative analysis of nucleic acid according to claim 1, wherein: the pH indicator has obvious color change at alkaline pH and acidic pH, and is cresol red, neutral red, phenol red or cresol purple.
3. The method for quantitative analysis of nucleic acid according to claim 1, wherein: the intelligent device is an intelligent mobile phone, a tablet personal computer or an intelligent bracelet with a shooting function.
4. An mRNA quantitative analysis application based on an intelligent device assisted nucleic acid quantitative analysis method, based on the nucleic acid quantitative analysis method of one of the above claims 1 to 3, characterized in that the mRNA quantitative analysis application comprises:
s1: constructing a pH-sensitive, mRNA-initiated, ligation-dependent isothermal nucleic acid amplification reaction system;
s2: at different reaction times, shooting and recording the color of the amplification reaction by using intelligent equipment;
s3: acquiring RGB color information of the picture in S2 through an image processing App;
s4: constructing a linear relation between the RGB value in the color information and the nucleic acid concentration, wherein the linear correlation equation is as follows:
G/(R+G+B)＝0.5078+0.0166lgCe13a2(M),[100pM-100fM]；
G/(R+G+B)＝0.7222+0.0331lgCe13a2(M)[100fM-100aM]；
s5: based on the linear relationship in S4, mRNA in the sample to be determined is quantified using an intelligent device.
5. The use of mRNA quantitation according to claim 4, wherein: the S1 construction of the isothermal nucleic acid amplification reaction system comprises the following steps:
s11: the detection recognition area of the probe SLP-e13 and the detection recognition area of the probe SLP-a2 are respectively hybridized with mRNA in the system specifically;
s12: using mRNA as a template, and connecting the probe SLP-e13 and the probe SLP-a2 by SplintR ligase to form double-stem ring structure DNA;
s13: and (3) initiating an amplification reaction by taking the DNA with the double-stem-loop structure as an initial template of the loop-mediated isothermal amplification reaction.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910538333.2A CN110265089B (en) | 2019-06-20 | 2019-06-20 | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof |
| PCT/CN2020/091831 WO2020253464A1 (en) | 2019-06-20 | 2020-05-22 | Nucleic acid quantitative analysis method based on intelligent device assistance and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910538333.2A CN110265089B (en) | 2019-06-20 | 2019-06-20 | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110265089A CN110265089A (en) | 2019-09-20 |
| CN110265089B true CN110265089B (en) | 2021-11-09 |
Family
ID=67919877
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910538333.2A Active CN110265089B (en) | 2019-06-20 | 2019-06-20 | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110265089B (en) |
| WO (1) | WO2020253464A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110265089B (en) * | 2019-06-20 | 2021-11-09 | 北京科技大学 | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof |
| CN113203730B (en) * | 2021-04-07 | 2022-06-21 | 上海大学 | Method for detecting trivalent chromium ions based on smart phone |
| CN113337584A (en) * | 2021-07-05 | 2021-09-03 | 清华大学深圳国际研究生院 | pH-sensitive nucleic acid quantitative detection method and device |
| CN113658635B (en) * | 2021-08-24 | 2023-09-29 | 北京诺禾致源科技股份有限公司 | Automatic determination method and device for nucleic acid detection result and application of automatic determination method and device |
| CN114002208B (en) * | 2021-10-26 | 2022-12-02 | 清华大学 | Paper-based sensor and mobile phone small program-based portable colorimetric detection method for heavy metal ions |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803062A (en) * | 2016-03-25 | 2016-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method |
| CN107541462A (en) * | 2017-09-13 | 2018-01-05 | 北京理工大学 | One kind is used for the system and application process of nucleic acid purification, amplification and genetic test |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120225491A1 (en) * | 2010-12-30 | 2012-09-06 | Ayal Ram | Portable detection devices and methods for detection of biomarkers and other analytes |
| CN105039321B (en) * | 2015-07-01 | 2018-09-25 | 中山大学 | A kind of modified constant temperature exponential amplification techniques and its application in microRNA detections |
| US10866184B2 (en) * | 2015-11-06 | 2020-12-15 | California Institute Of Technology | Devices and methods for direct visual detection and readout of single nucleic acid molecules |
| CN106706534A (en) * | 2016-12-30 | 2017-05-24 | 首都师范大学 | Method for detecting proteins based on combination of colorimetric array sensor and mobile phone |
| CN107384769B (en) * | 2017-08-24 | 2021-04-09 | 李峰 | Paper-based nucleic acid detection device and application thereof |
| CN107917905B (en) * | 2017-12-29 | 2020-11-20 | 山东师范大学 | Ratio type luminosity analysis device based on intelligent terminal and detection method thereof |
| CN110265089B (en) * | 2019-06-20 | 2021-11-09 | 北京科技大学 | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof |
-
2019
- 2019-06-20 CN CN201910538333.2A patent/CN110265089B/en active Active
-
2020
- 2020-05-22 WO PCT/CN2020/091831 patent/WO2020253464A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105803062A (en) * | 2016-03-25 | 2016-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Nucleic acid (DNA/RNA) real-time constant-temperature gene amplification detection method |
| CN107541462A (en) * | 2017-09-13 | 2018-01-05 | 北京理工大学 | One kind is used for the system and application process of nucleic acid purification, amplification and genetic test |
Non-Patent Citations (2)
| Title |
|---|
| "基于贵金属纳米颗粒散射谱编码的生物分子数字化检测";李国花;《中国博士学位论文全文数据库 工程科技Ⅰ辑》;20190215(第 02 期);第20，93页 * |
| "微流控等温扩增核酸定性与定量检测新方法研究";夏赟;《中国博士学位论文全文数据库 基础科学辑》;20170815(第 08 期);第121页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110265089A (en) | 2019-09-20 |
| WO2020253464A1 (en) | 2020-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110265089B (en) | Nucleic acid quantitative analysis method based on assistance of intelligent equipment and application thereof | |
| Kang et al. | Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example | |
| Varona et al. | Visual detection of single-nucleotide polymorphisms using molecular beacon loop-mediated isothermal amplification with centrifuge-free DNA extraction | |
| CA2884246C (en) | Multiplex pyrosequencing using non-interfering noise-canceling polynucleotide identification tags | |
| CA2905410A1 (en) | Systems and methods for detection of genomic copy number changes | |
| US20110151461A1 (en) | Detection algorithm for pcr assay | |
| CN106257989B (en) | NGS system controls and methods involving the same | |
| CN110317861B (en) | Kit for detecting pathogen | |
| KR20230024417A (en) | Systems and methods for analyzing biological samples | |
| CN107523624B (en) | Multi-cross isothermal amplification method combining AUDG and self-avoiding molecule recognition system | |
| US20180223381A1 (en) | Diagnostic methods and compositions | |
| CN106591475B (en) | General probe gene detection method, probe and application of probe | |
| US20180087096A1 (en) | Gene mutation detection method and fluorescence-labeled oligonucleotide used in same | |
| US20210292817A1 (en) | Methods for quantitation of analytes in multiplexed biochemical reactions | |
| CN110616261A (en) | Kit and method for detecting EGFR gene T790M mutation | |
| EP3966346B1 (en) | Combined solution phase and solid phase dna amplification | |
| WO2024015999A1 (en) | Methods, systems and compositions for detection of multiple analytes | |
| CN107557440B (en) | Loop-mediated isothermal amplification method combining AUDG and self-avoiding molecule recognition system | |
| CN107604085B (en) | LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for ureaplasma parvum | |
| CN117210540A (en) | Gene detection method based on three-way nucleic acid coded lock | |
| Pumford et al. | Developments in integrating nucleic acid isothermal amplification and detection systems for | |
| JP2017018011A (en) | Detection kit and detection method | |
| WO2023014898A1 (en) | Methods, systems and compositions for detection of multiple analytes | |
| CN117417986A (en) | Method for rapidly detecting target nucleic acid sequence based on 5-Br-PAPS | |
| CN112592966A (en) | Kit for rapidly detecting EGFR gene drug resistance mutation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |